Diagnostic and Staging Value of Serum Angiotensin-Converting Enzyme in Sarcoidosis

Objective. To investigate the diagnostic and staging value of serum angiotensin-converting enzyme (sACE) in sarcoidosis. Methods. Patients with suspected sarcoidosis treated in the Department of Pulmonary and Critical Care Medicine of the China-Japan Friendship Hospital from 2010 to 2020 were included. The data of sACE, erythrocyte sedimentation rate (ESR), complete blood count (CBC), lung function, bronchoalveolar lavage, and biopsy were collected. The differences between the sarcoidosis group and the nonsarcoidosis group and between different stages of sarcoidosis were compared. The receiver operating characteristic (ROC) curve analysis was used for the diagnostic test of sACE in sarcoidosis. Results. A total of 84 cases with suspected sarcoidosis were included, among which 70 cases were confirmed to be sarcoidosis by biopsy. The mean value of sACE in sarcoidosis patients was 56:61 ± 30:80U/L, which was significantly higher than that in nonsarcoidosis patients (28:07 ± 14:11U/L, P = 0:001). The level of sACE in sarcoidosis patients with peripheral superficial lymph nodes and multiple system involvement was significantly higher than that in intrathoracic sarcoidosis patients (P = 0:009); the percentage of lymphocytes in bronchoalveolar lavage fluid (BALF) of sarcoidosis patients was 45:39 ± 22:87%, which was significantly higher than that of nonsarcoidosis patients (P < 0:001). There was no correlation between sACE and ESR (correlation coefficient = −0:167). According to ROC curve analysis, when sACE ≥ 44:0U/L, the sensitivity of sarcoidosis diagnosis was 61.4%, the specificity was 92.9%, and the AUC was 0.819. Conclusion. sACE has a good specificity in the diagnosis of sarcoidosis. sACE values in patients with sarcoidosis with systemic involvement were higher than those with simple intrathoracic sarcoidosis.


Background
Sarcoidosis is a multisystemic granulomatous disease of unknown etiology. Pulmonary involvement is most frequent in sarcoidosis patients, while extrathoracic involvement occurs in only 30% of cases [1][2][3][4]. It has been reported that the prevalence and annual incidence of the disease have been on the rise in recent years [5]. In addition, mortality is higher in patients with sarcoidosis, especially those with comorbidities [6]. The clinical manifestations of sarcoidosis are variable and nonspecific, which makes it difficult to eliminate alternative diagnosis such as central lung cancer, hilar lymph node tuberculosis, and lymphoma, leading to misdiagnosis in clinical practice. A definitive diagnostic test is mainly made based on histopathology and adequate differential diagnosis [7]. It has been reported that while some patients with sarcoidosis experience spontaneous remission, others suffer from impaired quality of life and may incur higher medical costs as the disease progresses [8]. Therefore, exploring new methods for diagnosis and staging prediction of sarcoidosis is of great practical significance for improving the prognosis and quality of life of patients with sarcoidosis, which is also the significance of this study.
At present, increasing research results indicate that serum markers are of great value in disease diagnosis and prognosis prediction, with advantages of simple sample collection, minimal invasiveness, low cost, high sensitivity, and specificity [9,10]. Clinically, more and more serum markers have been applied in the diagnosis and prognosis of sarcoidosis [11]. For example, serum chemokine (C-X-C motif) ligand 9 (CXCL9) is associated with pulmonary outcome and disease burden in patients with sarcoidosis and can be used to assist in the identification of organ involvement, respiratory symptom severity, and pulmonary dysfunction in patients with sarcoidosis [12]. Miyata et al. [13] pointed out in their study that serum-soluble interleukin-2 receptor (SIL-2R) could assist in endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), in the diagnosis of sarcoidosis, helping to improve the diagnosis rate. Serum angiotensin-converting enzyme (sACE) is a converting enzyme that can convert β-amyloid peptide (Aβ) 42 into Aβ40 in the human brain, and its conversion activity is mediated by two catalytic domains [14]. sACE, which is abnormally upregulated in patients with sarcoidosis and significantly higher than that in healthy controls, has been shown to be a serum biomarker for the diagnosis of sarcoidosis or monitoring of disease activity [15]. This survey summarized suspected sarcoidosis cases admitted to the Department of Pulmonary and Critical Care Medicine of the China-Japan Friendship Hospital from 2010 to 2020, aiming to analyze the role of sACE in the diagnosis and evaluation of sarcoidosis.

2.1.
Participants. This study prospectively included patients with suspected sarcoidosis hospitalized in the Department of Pulmonary and Critical Care Medicine of the China-Japan Friendship Hospital between 2010 and 2020. A total of 84 cases of suspected sarcoidosis were included, 70 cases of which were histopathologically confirmed. Among them, 9 cases were confirmed as lymphatic tuberculosis, 2 cases as malignancy, and 3 cases as other diseases that cause enlargement of mediastinal and hilar lymph nodes. According to the histopathological diagnosis, 70 patients with sarcoidosis were assigned to the sarcoidosis group, and the remaining 14 patients were assigned to the nonsarcoidosis group. Almost all the cases presented with cough, expectoration, chest tenderness, or shortness of breath, while fever as well as fatigue and weight loss were observed in 18 and 19 cases, respectively.
Patients meeting one of the following criteria were considered suspected sarcoidosis: (1) chest radiograph or computed tomography showing bilateral or unilateral hilar or mediastinal lymphadenopathy, with or without pulmonary opacities; (2) on the basis of (1), patients presented with respiratory symptoms such as cough, dyspnea, and chest pain, accompanied by fatigue, malaise, fever, or weight loss; and (3) other conditions that cannot exclude sarcoidosis as confirmed by a respiratory physician.
The diagnostic criteria of sarcoidosis were based on the following: (1) consistent clinical and radiographic findings, (2) histopathologic detection of nonnecrotizing granulomas, and (3) exclusion of other diseases.
Sarcoidosis patients were staged for the disease according to the Scadding system of staging [7]: stage I: bilateral hilar lymphadenopathy (BHL); stage II: pulmonary infiltration with BHL; stage III: pulmonary infiltration without BHL; and stage IV: pulmonary fibrosis. According to the disease staging, patients in the sarcoidosis group were further divided into the stage I group (n = 14, 20.00%), stage II group (n = 50, 71.43%), and stage III group (n = 6, 8.57%), with no stage IV patients. Furthermore, sarcoidosis patients were assigned to the intrathoracic sarcoidosis group (n = 19, 27.14%), sarcoidosis-involved intrathoracic organ and peripheral lymph node group (n = 39, 55.71%), and multisystemic sarcoidosis group (n = 12, 17.14%) based on the location of nodular involvement. Among extrathoracic sarcoidosis, there were 2 cases of skin involvement, 4 cases of hepatic involvement, 3 cases of splenic involvement, 3 cases of ocular involvement, and 1 case of cardiac involvement.
Written informed consent was obtained prior to data collection. The study was approved by the Ethics Committee of China-Japan Friendship Hospital.

Laboratory Testing
(1) sACE: the peripheral blood serum was collected from patients to detect sACE using an ultraviolet spectrophotometer.
(2) Complete blood count (CBC): CBC, including white blood cell count (WBC), lymphocytes, and hemoglobins, as well as the level of C-reactive protein (CRP), was routinely measured on the collected peripheral blood through a blood analyzer (Shanghai Jumu Medical Equipment Co., Ltd., China, a160).

Histopathology.
Biopsy specimens were obtained via EBUS-TBNA, transbronchial lung biopsy (TBLB), CTguided percutaneous fine-needle aspiration, video-assisted thoracoscopic surgery, peripheral lymph node biopsy, skin biopsy, etc. Of the above various sample collection methods, EBUS-TBNA and/or TBLB was performed in 50 cases, skin biopsy or peripheral lymph node biopsy was performed in 13 cases, CT-guided percutaneous fine-needle aspiration was performed in 2 cases, and video-assisted thoracoscopic surgery was performed in 5 cases.

Statistical
Analysis. All data were analyzed by statistical software SPSS 21.0. The data were presented in the format of mean ± SD. Unpaired t test and analysis of variance were used to compare the differences in variables between groups. Count data, represented as a constituent ratio, was compared by the chi-square test. Pearson's correlation analysis was used for correlation analysis between two groups. The diagnostic test of sACE in sarcoidosis was performed by receiver operating characteristic (ROC) curve analysis. Differences with P values < 0.05 were considered statistically significant.

Demographic Characteristics and Pathological Data
Analysis. Among the 70 sarcoidosis cases, 56 cases (80.00%) were female. The mean age of the total sarcoidosis cases was 55:94 ± 11:83 years, with 52 cases (74.28%) presenting respiratory symptoms. The mean value of sACE in sarcoidosis patients was 56:61 ± 30:80 U/L, which was significantly higher than that in nonsarcoidosis patients (28:07 ± 14:11 U/L, P = 0:001). The percentages of lymphocytes and neutrophils in BALF of sarcoidosis patients were significantly higher than those of nonsarcoidosis patients (P < 0:001). Although 30 (42.85%) sarcoidosis patients presented with elevated ESR, there was no statistical difference in ESR level between sarcoidosis and nonsarcoidosis patients nor were there any significant differences in age and results of CBC and PFT between sarcoidosis and nonsarcoidosis patients. Forty-six sarcoidosis patients underwent tuberculin skin test (TST), returning 38 (82.61%) negative, 4 (8.70%) weak positive, and 2 (4.35%) positive results. Twenty cases exhibited abnormal PFT results, with 5 cases showing restrictive ven-tilatory defect, 14 cases showing obstructive ventilatory defect, and 1 case showing mixed ventilatory defect. The damage of PFT was mild in sarcoidosis patients.

Comparison between Sarcoidosis of Different
Types of System Involvement. The sACE level in patients with intrathoracic sarcoidosis was 47:05 ± 23:66 U/L, the sACE level in sarcoidosis patients involved both intrathoracic organs and peripheral lymph nodes was 56:47 ± 29:68 U/L, and the sACE level in sarcoidosis patients with multisystemic involvement was 64:94 ± 39:72 U/L. The level of sACE in sarcoidosis patients with peripheral superficial lymph nodes and multisystemic involvement was significantly higher than that in intrathoracic sarcoidosis patients (P = 0:009), while there was no significant difference in age, gender, clinical manifestations, CBC, ESR, and PFT among them. Data are shown in Table 2.

Diagnostic Evaluation of sACE in Sarcoidosis.
According to ROC curve analysis, when sACE ≥ 35:4 U/L, the sensitivity for diagnosing sarcoidosis was 78.6%, the specificity was 78.6%, the positive predictive value was 94.6%, and the negative predictive value was 42.3%. When sACE ≥ 44:0 U/L, the sensitivity for diagnosing sarcoidosis was 61.4%, the specificity was 92.9%, and the area under the curve (AUC) was 0.819. Thus, sACE ≥ 44:0 U/L may be an ideal cut-off value in sarcoidosis diagnosis. The ROC curve is shown in Figure 1.

Correlation Analysis between sACE and ESR.
There was no correlation between sACE and ESR ( Figure 2).

Discussion
Sarcoidosis is a multisystemic granulomatous disease with the common respiratory symptoms including cough, dyspnea, and chest pain, often accompanied by fatigue, malaise, fever, and weight loss [16]. Sarcoidosis usually has insidious 3 Computational and Mathematical Methods in Medicine onset with mild and nonspecific symptoms, which can easily lead to misdiagnosis and missed diagnosis. The results of this study showed that sACE levels were significantly elevated in patients with sarcoidosis, which was associated with disease activity and extent. The ROC analysis revealed that the optimal critical value of sACE for the diagnosis of sarcoidosis was 44.0 U/L. Although the sensitivity was 61.4%, the specificity was as high as 92.9%, indicating that sACE still has important clinical value in the diagnosis of sarcoidosis.
Of the 70 sarcoidosis cases in this study, 52 cases (74.28%) presented with varying degrees of respiratory symptoms such as cough, expectoration, chest distress, and shortness of breath. Extrathoracic involvement can occur  in some sarcoidosis patients, which mainly involves the skin, eye, liver, spleen, and cardiac system, presenting as fever, weight loss, and fatigue. In this study, the peak age of onset is middle-aged, with a predilection for women, which is consistent with the previous report [2]. Characteristic PFTs of sarcoidosis will reveal restrictive ventilatory defects (reduced FVC and TLC) with decreased DLCO; however, the normal pattern of PFTs is not rare in clinical practice [17]. Over 90% of sarcoidosis patients have pulmonary involvement [18,19], so assessment of chest radiography is essential in sarcoidosis diagnosis. In this study, mediastinal lymphadenopathy (64 cases, 91.43%) and BHL (54 cases, 77.14%) were the main radiographic abnormalities of sarcoidosis. Although BHL is the typical radiographic abnormality of sarcoidosis [20], 5 sarcoidosis cases in this study exhibited unilateral lymphadenopathy, which were more likely to be misdiagnosed as tumor or tuberculosis. The final diagnosis of sarcoidosis is based on histopathology, so histopathology is strongly recommended in patients with suspected sarcoidosis. Peripheral lymph node biopsy or skin biopsy is a simple, safe, and reliable diagnostic approach. For patients without peripheral lymphadenopathy or skin involvement, EBUS-TBNA combined with TBLB is a relatively simple and safe diagnostic method with a high positive rate. In this survey, 50 cases underwent EBUS-TBNA and/or TBLB, and the histopathological diagnostic rate was 71.4%, which was similar to the positive rate of 88% reported internationally.
The exact etiology and pathogenesis of sarcoidosis remain unknown. In the diagnosis and prognosis evaluation of sarcoidosis, the ideal marker to indicate diagnostic approach and disease activity monitoring has not yet been discovered [21]. Elevated sACE can indicate disease activity  and systemic involvement, with some certain degree of specificity for the diagnosis of sarcoidosis. The main function of ACE is to convert angiotensin I to angiotensin II and inactivate bradykinin. ACE is mainly distributed in the epithelial cell and cysts of pulmonary capillary endothelia. ACE is also found in the epithelial or vascular endothelial cells of the liver, kidney, brain, eye, and small intestine. The level of sACE is closely correlated with pulmonary disease as abundant capillaries are distributed in the lung. sACE is elevated in 75% of untreated sarcoidosis patients [22]. The diagnostic value of sACE in other diseases has also been well confirmed. It is shown that sACE is significantly related to the infection mechanism of novel coronavirus (SARS-CoV-2) and can be used to diagnose patients with such infection [23]. sACE can also be used as a serum exosomal messenger RNA (mRNA) indicator to diagnose early diabetic nephropathy [24]. In this study, the level of sACE in sarcoidosis patients was significantly higher than that in nonsarcoidosis patients with hilar and/or mediastinal lymphadenopathy. sACE can be an indicator for differential diagnosis of lymphatic involvement in nonsarcoidosis patients. It is reported that ESR is a useful marker of sarcoidosis activity [25]. The level of ESR showed no significant difference between sarcoidosis and nonsarcoidosis patients, and there was no correlation between sACE and ESR, which indicates that sACE is better than ESR in reflecting disease activity. In addition, the BALF lymphocyte percentage was significantly higher in sarcoidosis patients, suggesting that the BALF lymphocyte percentage can be a sensitive indicator for the diagnosis of sarcoidosis, though with no correlation with disease severity.
Sarcoidosis can involve multiple systems with complex and varied clinical manifestations. Over 90% of sarcoidosis patients have pulmonary involvement. Although extrathoracic presentations as the initial symptoms of sarcoidosis are rare, accounting for 23% of sarcoidosis inpatients, they can be the main clinical manifestations and characteristics in some patients with sarcoidosis. According to a survey of 22 sarcoidosis cases whose initial symptoms were extrathoracic symptoms, 6 cases were identified as peripheral lymph-adenopathy, 5 cases as skin involvement, 2 cases as ocular involvement, 3 cases as parotid gland sarcoidosis, 3 cases as arthritis, 1 case as cardiac sarcoidosis, 1 case as hepatic and splenic enlargement, and 1 case as neurologic sarcoidosis. Sarcoidosis has varying clinical manifestations and characteristics, which are related to race, staging, systemic involvement, and granulomas activity. The results of this study indicated that the sACE level was significantly higher in extrathoracic sarcoidosis. sACE levels reflect the varying degrees of granuloma, rather than pure pulmonary involvement, and are correlated with disease activity and extent. The level of sACE tends to be higher when sarcoidosis is active. Thus, sACE can indicate extrathoracic involvement in sarcoidosis.
In this study, 44.0 U/L of sACE was defined as the optimal diagnostic cut-off for sarcoidosis diagnosis. We used 44.0 U/L as the diagnostic cut-off and histopathology as the golden diagnostic criterion to make a diagnosis in 84 cases. The results showed that although the sensitivity of sACE ≥ 44:0 U/L for sarcoidosis diagnosis was 61.4%, the specificity of 92.9% was high and ideal. According to data from Judson [26], the sensitivity of sACE for sarcoidosis was 57% and the specificity was 90%, which was similar to our conclusions. The poor sensitivity may be due to a variety of factors affecting the regulation of ACE expression, such as gene polymorphism and environmental factors [27]. Nevertheless, sACE still has significant clinical value in the diagnosis of sarcoidosis, especially in terms of diagnostic specificity. The novelty of this study is that we evaluated the clinicopathological features of patients with sarcoidosis from the aspects of demographic characteristics, chest X-rays, histopathology, laboratory tests, and bronchoscopy and used the serum marker sACE as a breakthrough to analyze its manifestations in different systems and different clinical stages of sarcoidosis. Finally, the ROC analysis confirmed that sACE is an effective serum index for the diagnosis of sarcoidosis, which has certain clinical reference value for the screening and staging prediction of sarcoidosis.
sACE is an important diagnostic approach for sarcoidosis diagnosis. However, the normal range of sACE should be revised based on analysis of large sample data from multicenter investigations to improve the diagnostic efficiency of sarcoidosis. In addition, the high level of sACE in sarcoidosis patients with extrathoracic involvement suggests that sACE can be used as an important indicator for further screening and assessment of extrapulmonary involvement.

Data Availability
The labeled dataset used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.

Authors' Contributions
Wenqiao Wang and Yue Ma contribute equally to this work.   Computational and Mathematical Methods in Medicine